Mechanism of drug resistance and reversal with ligustra-zine and cyclosporin A in cisplatin--inducedhuman epithelial ovarian cancer resistant cell line 3Ao/cDDP

Objective: To investigate the mechanism of resistance and reversal effect of ligustrazine and cyclosporin A in cisplatin--induced multidrug resistance ovarian cancer cell line 3Ao/cDDP. Methods: Using the corresponding dose calculated from clinical chemotherapy at 30 mg cisplatin per cycle, we established 3Ao/cDDP with 3Ao exposed at regular intervals and repeatedly to high-level concentration of cisplatin at 10 mg/ml for 24 hours each time. Expressions of LRP, MRP, P-gp, GSTp and TopoII were quantitatively detected with FCM. For drug resistance reversal, cyclosporin A and ligustrazine were administered singly or in combination at the maximal dose without cytotoxicity. Inhibition rates were determined by MTT assay. Results: 3Ao/cDDP was established after 4.5 months, with resistance factor 1.6 which was similar to clinical resistance degree. Low expression levels of MRP and P-gp were found in both 3Ao and 3Ao/cDDP (P>0.05), and LRP and GSTp expression levels in 3Ao/cDDP were significantly higher than those in 3Ao (P<0.005 and P<0.05, respectively), and TopoII in 3Ao/cDDP was significantly lower vs 3Ao (P<0.05). The inhibition rate of cDDP was 20.807±0.015%, cDDP plus ligustrazine 27.421±0.07% (P>0.05 vs cDDP), cDDP plus cyclosporin A 49.635±0.021% (P<0.01 vs cDDP), and cDDP plus ligustrazine and cyclosporin A 58.861±0.014% (P<0.01 vs cDDP). Conclusions: 3Ao/cDDP, induced by cisplatin and established by imitating the characteristics of clinical chemotherapy for epithelial ovarian cancer, was an ideal model for investigation of cisplatin resistance in vitro. Cisplatin resistance in 3Ao/cDDP could be accounted for by higher LRP, GSTp and lower TopoII expression and was not associated with MRP or P-gp. Ligustrazine had no significant reversal effect on cisplatin resistance, but cyclosporin A could reverse the resistance effectively.

Construction of Three-dimensional In Vitro Culture Model of Ovarian Carcinoma and the Study of Its Multicellular Drug Resistance

To explore the role and possible mechanism of apoptosis and caspase-3 activity in the development of multicellular drug resistance of ovary cancer. Ovarian cancer cell A2780 multicellular spheroids （MCS） were obtained from three-dimensional culture. Drug sensitivity of monolayer cells （MC） and MCS were respectively tested by MTT staining and cytometry. The apoptosis of MC and MCS were determined by the flow cytometry （FCM）. The expression of bcl-2 and caspase-3 in A2780/MC and A2780/MCS were detected by using Western blot and caspase-3 assay kit, A2780/MC was compacted into mass after 2 days in three-dimensional cell culture model, and MCS had more than two layers of cells growing within 5 days. Compared with A2780/MC, A2780/MCS were more resistant to the anticancer drug, and the apoptosis rate was significantly lower than those of A2780/MC, The activity of caspase-3 in A2780/MCS was significantly lower than the A2780/MC. But the expression of bcl-2 in A2780/MCS was significantly higher than that in A2780/MC. It was suggested that the drug resistance of MCS might be associated with the overexpression of anti-apoptosis protein bcl-2 and the down-regulation of caspase-3 activity.

Effects of Taxotere on invasive potential and multidrug resistance phenotype in pancreatic carcinoma cell line SUIT-2

INTRODUCTIONDevelopment of drug-resistance to chemotherapyand subsequent metastasis of tumor are primarilyresponsible for treatment failure and the death fromcancer. There have been many previous studies onthe relationship between expression of multidrugresistance (MDR) phenotype P-glycoprotein (P-gp)and the malignant properties of tumors, but theresults are often conflicting[1-8]. The difference intumor types or MDR phenotype induced by specificagents might account for this discrepancy. Taxotere(TXT), a member of the family of taxanes, hasantitumor activity through its effect of promotingthe polymerization of tubulin[9,10].

Bioinformatics Identification of ZNFs/LINC00520/miR-181d/BCL2 Axis as a Novel Network in Cisplatin-Resistant Lung Adenocarcinoma Cells

Background: Resistance to cisplatin (DDP) leads to poor prognosis in patients with Lung Adenocarcinoma (LUAD) and limits its clinical application. It has been confirmed that autophagy promotes chemoresistance and, therefore, novel strategies to reverse chemoresistance by regulating autophagy are desperately needed. Methods: The differentially expressed lncRNAs (DElncRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) between A549 and A549/DDP cell lines were identified using the limma package in R, after gene expression profiles were obtained from Gene Expression Omnibus (GEO) database. By combining Autophagy-Related Genes (ARGs) from Human Autophagy Database (HADb), the interactions lncRNA-miRNAs and the interactions miRNAs-mRNAs respectively predicted by miRcode and miRDB/Targetscan database, the autophagy-related ceRNA network was constructed. Then, extraction of ceRNA subnetwork and Cox regression analyses were performed. A prognosis-related ceRNA subnetwork was constructed, and the upstream Transcription Factors (TFs) regulating lncRNAs were predicted by the JASPAR database. Finally, the expression patterns of candidate genes were further verified by quantitative real-time polymerase chain reaction (qRT-PCR) experiments. Results: A total of 3179 DEmRNAs, 180 DEmiRNAs, and 160 DElncRNAs were identified, and 35 DEmRNAs were contained in the HADb. Based on the ceRNA hypothesis, we established a ceRNA network, including 10 autophagy-related DEmRNAs, 9 DEmiRNAs, and 14 DElncRNAs. Then, LINC00520, miR-181d, and BCL2 were identified to construct a risk score model, which was confirmed to be a well-predicting prognostic factor. Furthermore, 5 TF ZNF family members were predicted to regulate LINC00520, whereas the RT-PCR results showed that the 5 ZNFs were consistent with the bioinformatics analysis. Finally, a ZNF regulatory LINC00520/miR-181d/BCL2 ceRNA subnetwork was constructed. Conclusions: An ZNFs/LINC00520/miR-181d/BCL2 axis as a novel network in DDP-resistant LUAD has been constructed successfully, which may provide potential therapeutic targets for LUAD.

MULTICELLULAR-MEDIATED RESISTANCE TO CISPLATIN AND TAXOL IN HUMAN OVARIAN CANCER SK-OV-3IP1 MULTICELLULAR AGGREGATES

Objective: To investigate the chemosensitivity of ovarian cancer SK-OV-3ip1 multicellular aggregates (MCA) to cisplatin and taxol and to explore the possible mechanisms. Methods: Liquid overlay system was employed to obtain MCA. We detected the resistance using trypan blue exclusion testing, clonogenic assay, cell cycle profiles and apoptosis with flow cytometry (FCM). Results: After cisplatin exposure, MCA cells showed nearly equal cell viability with monolayer cells (P=0.05). After 40μM cisplatin exposure for 12 h, no clone (≥50 cells) was formed, but more viable cells attached to the bottom of 24-well plate in MCA group than monolayer. Furthermore, apoptosis rate and cell cycle profiles with FCM had no significant change between MCA and monolayer cells. After taxol exposure, however, trypan blue exclusion testing demonstrated higher cell viability in MCA cells (P=0.003) and higher clone formation rate in 100-cell group than monolayer cells (0.01<P<0.025). No significant difference was found in 50-cell or 200-cell group but more viable cells in MCA group were observed. Taxol exposure caused significantly decreased apoptosis rate in MCA cells than monolayer cells (P=0.012). Taxol induced significant cell arrest at G2-M phase in monolayer cells (P=0.001), but abrogation of G2-M arrest was observed in MCA cells (P=0.002). Conclusion: Compared with monolayer cells, MCA cells from the same SK-OV-3ip1 cell line appear to be more resistant to taxol but not to cisplatin. Cell cycle redistribution and multicellular-mediated inhibition of apoptosis can partially account for the resistance.

SCREENING OF DRUG RESISTANCE-RELATED GENES FROM HUMAN OVARIAN CANCER CELL LINE OC3/ADR BY DD-PCR

Objective: To screen novel genes related to adriamycin (Adr) resistance from human ovarian cancer resistance cell line OC3/Adr. Methods: Multidrug resistant ovarian cancer cell line OC3/Adr was induced by intermittent treatment of the human parent cell line OC3 with high concentration Adr. The difference of gene expression was screened by using different display analysis to the acquired Adr-resistance subline OC3/Adr and its parent cell line OC3. Results: OC3/Adr cell line was obtained which was more resistance to Adr than the parent cell line OC3 with the resistance index (RI) of 15.4. The OC3/Adr cell line also showed cross-resistance to other anti-cancer drugs (VP16, CDDP,5FU). It grew slowly and exhibited changes of cell cycle. A number of differentially expressed ESTs (Expressed Sequence Tags, ESTs) were identified at mRNA level between the OC3/Adr and OC3. Four of 18 different ESTs were sequenced. The 431/432 base pair S1 was homologous to human sperm zona pellucida binding protein, while the other two ESTs, S3 and S4, were new gene segments, which were registered to GenBank with the number of AF 117656 and AF 126507 respectively. Particularly, the expression of S2 sequence increased in all the drug-resistance cell lines and S3 sequence overexpressed in human ovarian cancer tissues as compared with benign ovarian tumors. Conclusion: Drug resistance induced by Adr in ovarian cancer OC3/Adr is involved with changes of multiple gene expressions.

Molecular mechanisms of cisplatin resistance in ovarian cancer

Ovarian cancer is one of the most common malignant tumors of the female repro-ductive system.The majority of patients with advanced ovarian cancer are mainly treated with cisplatin-based chemotherapy.As the most widely used first-line anti-neoplastic drug,cisplatin produces therapeutic effects through multiple mechanisms.However,during clinical treat-ment,cisplatin resistance has gradually emerged,representing a challenge for patient outcome improvement.The mechanism of cisplatin resistance,while known to be complex and involve many processes,remains unclear.We hope to provide a new direction for pre-clin-ical and clinical studies through this review on the mechanism of ovarian cancer cisplatin resis-tance and methods to overcome drug resistance.

Taxotere resistance in SUIT Taxotere resistance in pancreatic carcinoma cell line SUIT 2 and its sublines

AIM: To investigate the specific mechanisms of intrinsic and acquired resistance to taxotere (TXT) in pancreatic adenocarcinoma (PAC). METHODS: MTT assay was used to detect the sensitivity of PAC cell line SUIT-2 and its sublines (S-007, S-013, S-020, S-028 and TXT selected SUIT-2 cell line, S2/TXT) to TXT. Mdr1 (P-gp), multidrug resistance associated protein (MRP), lung resistance protein (LRP) and beta-tubulin isotype gene expressions were detected by RT-PCR. The functionality of P-gp and MRP was tested using their specific blocker verapamil (Ver) and indomethacin (IMC), respectively. The transporter activity of P-gp was also confirmed by Rhodamine 123 accumulation assay. RESULTS: S-020 and S2/TXT were found to be significantly resistant to TXT(19 and 9.5-fold to their parental cell line SUIT-2, respectively). RT-PCR demonstrated strong expression of Mdr1 in these two cell lines, but weaker expression or no expression in other cells lines. MRP and LRP expressions were found in most of these cell lines. The TXT-resistance in S2-020 and S2/TXT could be reversed almost completely by Ver, but not by IMC. Flow cytometry showed that Ver increased the accumulation of Rhodamine-123 in these two cell lines. Compared with S-020 and SUIT-2, the levels of beta-tubulin isotype II, III expressions in S-2/TXT were increased remarkably. CONCLUSION: The both intrinsic and acquired TXT-related drug resistance in these PAC cell lines is mainly mediated by P-gp, but had no relationship to MRP and LRP expressions. The increases of beta-tubulin isotype II, III might be collateral changes that occur when the SUIT-2 cells are treated with TXT.

Experimental study on antitumor effect of arsenic trioxide in combination with cisplatin or doxorubicin on hepatocellular carcinoma

INTRODUCTIONThe main component of a traditional Chinese drug 'Pishuang'. arsenic trioxide (As2O3), has obviously selective anti-tumor effect on human hepatocellular carcinoma (HCC)in both in vitro and in vivo studies[1-5]. Due to limited effectiveness when any anti-carcinogen is used alone and obviously increased toxicity when the dose is raised, there is no exception for As2O3. Furthermore, combined chemotherapy contributes to improve therapeutic effectiveness, disperse toxicity and surmount drug-resistance,in which the combination of traditional Chinese and modern medicine has more advantages and characteristics. As a result,we made an experimental study on anti-tumor effect of As2O3in combination with cisplantin (PDD) or doxorubicin (ADM)on HCC. to investigate the possibility of AS2O3 in combination with PDD or ADM and nature of interaction between them,and to provide experimental basis for clinical application.

Surgery in platinum-resistant recurrent epithelial ovarian carcinoma

BACKGROUND Ovarian cancer is one of the three most common malignant tumors of the female reproductive tract and ranks first in terms of mortality among gynecological tumors.Epithelial ovarian carcinoma(EOC)is the most common ovarian malignancy,accounting for 90%of all primary ovarian tumors.The clinical value of cytoreductive surgery in patients with platinum-resistant recurrent EOC remains largely unclear.AIM To evaluate the feasibility of secondary cytoreductive surgery for treating platinum-resistant recurrent EOC.METHODS This was a retrospective study of the clinical data of patients with platinumresistant EOC admitted to the Cancer Hospital of the University of Chinese Academy of Sciences between September 2012 and June 2018.Patient baseline data were obtained from clinical records.Routine follow-up of disease progression was performed as follows.CA125 assessment and physical examination were performed every 3 wk during treatment,including gynecological examination.Imaging assessment was carried out every 12 wk by B-mode ultrasound,computed tomography,or magnetic resonance imaging.The primary outcome was progression-free survival(PFS).Secondary outcomes included overall survival(OS),chemotherapy-free interval(CFI),and complications.Follow-up ended on April 15,2019.RESULTS A total of 38 patients were included.R0 resection was achieved in 25(65.8%) patients and R1/2 in 13 (34.2%). Twenty-five (65.8%) patients required organ resection. Nine(23.7%) patients had operative complications, 36 (94.7%) received chemotherapy, and five (13.2%)had targeted therapy. Median PFS and OS were 10 (95%CI: 8.27-11.73) months and 28 (95%CI:12.75-43.25) months, respectively;median CFI was 9 (95%CI: 8.06-9.94) months. R0 resection andpostoperative chemotherapy significantly prolonged PFS and OS (all P < 0.05), and R0 resectionalso significantly prolonged CFI (P < 0.05). Grade ≥ 3 complications were observed, includingrectovaginal fistula (n = 1), intestinal and urinary fistulas (n = 1), and renal failure-associated death(n = 1). Except for the patient who died after surgery, all other patients with complications weresuccessfully managed. Two patients developed intestinal obstruction and showed improvementafter conservative treatment.CONCLUSIONSecondary cytoreductive surgery is feasible for treating platinum-resistant recurrent EOC. Thesefindings provide important references for the selection of clinical therapeutic regimens.

Reversal of HCC Drug Resistance by Using Hammerhead Ribozymes against Multidrug Resistance 1 Gene

To reverse multidrug resistance（MDR） of HepG2 by anti-MDR1 hammerhead ribozyme, an anti-MDR1 hammerhead ribozyme was developed and delivered to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retroviral vector containing RNA polymerase Ⅲ promoter. The expression of mdrl/Pgp and Rz was detected in HepG2, HepG2 muhidrug-resistant cell line and HepG2 Rz-transfected cells by semi-quantitative RT-PCR and Western blot methods. Moreover, MTT assay was employed to detect the sensitivity of these ribozyme-transfected cells, and Rhodamine123 （Rh123） was used to test the function of Pgp. The Rz- transfected HepG2 cells became doxorubicin-sensitive, which was concomitant with the decreased MDR1 expression. The study showed that the retrovirus vector encoding the anti-MDR1 ribozyme may be applicable to the treatment of MDR cells.

Toxicity of Paclitaxel and Cisplatin in Combination for Advanced Ovarian Cancer

Objective:To evaluate the toxicity of paclitaxel and cisplatin combination in patients with advanced ovarian cancer.Methods:A retrospective review was performed on patients with stage Ⅲ or stage Ⅳ ovarian cancer treated in QiLu hospital between October 1996 and June 1999 Results:26 patients received adjuvant paclitaxel and cisplatin chemotherapy.The significant toxicity included:anemia 7/26(27%),thrombocytopenia 10/26(38%),neutropenia 19/26(73%),nausea or vomiting 24/26(92%) and neurotoxicity 11/26(42%).No patient delayed.No patient died during treatment.Conclusion:The dosages of primary paclitaxel and cisplatin chemotherapy is reasonably well tolerated for patients with stage Ⅲ or Ⅳ ovarian cancer.

肝癌靶向联合免疫治疗耐药后的二线治疗方案研究进展

近年来,靶向和免疫单药及联合治疗晚期肝癌的临床研究为一线用药方案选择提供了丰富的疗效与安全性证据。然而,对于肝癌二线治疗方案的选择,目前各项临床指南尚无统一意见,原因在于现有循证医学证据局限于索拉非尼失败后的选择,而对于新的一线方案,如靶向免疫联合治疗肝癌耐药后的二线治疗方案,依然缺乏高证据等级的临床试验结论。本文回顾了目前临床试验研究结果,根据药物作用的不同机制,对靶向免疫一线治疗耐药后肝癌二线治疗方案的研究进行了归纳,并系统总结近年研究进展。对于一线靶免联合治疗耐药的肝癌患者,靶向联合治疗、免疫双抗治疗均有望提高疗效、改善生存,未来还需更多前瞻性临床研究数据,为靶免联合治疗耐药的肝癌患者提供有效、安全的治疗方案。

卵巢癌化疗耐药预测模型的建立及效果评价

目的探讨卵巢癌患者术后化疗发生耐药的影响因素,构建预测模型并评价模型效能。方法收集经肿瘤细胞减灭术及化疗的407例卵巢癌患者的临床资料,至随访终点根据是否复发分为复发组363例和未复发组44例,其中复发组根据化疗耐药将其分为耐药组59例和敏感组304例。使用单因素分析和Lasso回归筛选变量,建立Logistic模型,用R软件建立列线图并进行评价。结果与未复发组比较,复发组年龄偏低,低分化比例及FIGO分期Ⅲ—Ⅳ期比例较高(P<0.05)。与敏感组比较,耐药组淋巴结增大、病理类型为非浆液性、FIGO分期Ⅲ—Ⅳ期比例、肿瘤组织免疫组化重组蛋白Ki-67(Ki-67)、蛋白53(P53)、血管内皮生长因子(VEGF)及肾母细胞瘤基因1(WT-1)阳性率较高,手术前后糖类抗原125(CA125)变化率、化疗前后罗马指数(绝经前)变化率及免疫组化蛋白16(P16)阳性率较低(P<0.05)。以Lasso回归筛选出的8个自变量进行Logistic回归,结果显示:术前全腹增强CT有淋巴结增大、病理类型为非浆液性、FIGO分期Ⅲ—Ⅳ期、免疫组化WT1、VEGF阳性,P16阴性是卵巢癌患者发生化疗耐药的独立危险因素。据此建立的列线图模型受试者工作特征曲线下面积为0.837(0.783~0.880),Hosmer-Lemeshow检验结果示模型拟合优度较好,校准曲线及临床决策曲线提示模型有较高的校准度及临床使用度。结论根据临床数据成功构建了卵巢癌化疗耐药Logistic模型,据此建立的列线图预测模型可有效评估卵巢癌患者发生化疗耐药的风险。

鸦胆子油乳对肺癌A549细胞顺铂化疗的增敏作用及其机制

目的观察鸦胆子油乳对肺癌A549细胞顺铂化疗的增敏作用并分析其机制。方法体外培养人肺癌细胞A549,MTT法检测鸦胆子油乳、顺铂对细胞生长的抑制率,计算鸦胆子油乳、顺铂对细胞的半数抑制浓度(IC_(50)),观察不同浓度(2.44、9.77、78.12μg/mL)鸦胆子油乳对顺铂IC_(50)的影响。将A549细胞分为对照组、鸦胆子油乳组、顺铂组及鸦胆子油乳+顺铂组,对照组不做处理正常培养细胞,鸦胆子油乳组、顺铂组及鸦胆子油乳+顺铂组分别给予鸦胆子油乳单药、顺铂单药、鸦胆子油乳及顺铂联合处理。采用荧光显微镜观察细胞形态及生长情况,流式细胞术检测细胞周期及细胞凋亡率,二氯荧光素二乙酸酯荧光探针染色检测细胞内ROS水平。结果顺铂IC_(50)随着鸦胆子油乳处理浓度的增高而下降(P均<0.05)。对照组细胞生长较好,细胞数量较多且形状为正常生长状态;顺铂组及鸦胆子油乳组细胞数量较对照组均减少,可见少量碎片;鸦胆子油乳+顺铂组细胞生长受抑制较明显,细胞数量最少,细胞形状不规则且细胞碎片增多。G_(0)/G_(1)期细胞比例鸦胆子油乳+顺铂组>顺铂组、鸦胆子油乳组>对照组,细胞凋亡率鸦胆子油乳+顺铂组>鸦胆子油乳组、顺铂组>对照组,细胞内ROS水平鸦胆子油乳+顺铂组>鸦胆子油乳组、顺铂组>对照组(P均<0.05)。结论鸦胆子油乳能够增加肺癌A549细胞对顺铂化疗的敏感性,其机制可能与阻滞肿瘤细胞于G_(0)/G_(1)期、增加活性氧产生从而促进细胞凋亡有关。

华蟾素调控PI3K/AKT通路逆转卵巢癌A2780/DDP细胞顺铂耐药的作用机制

目的研究华蟾素(CBG)对人卵巢癌细胞顺铂耐药的逆转作用和机制。方法A2780细胞株及其顺铂耐药细胞株A2780/DDP是临床常见的卵巢癌细胞,故选择这两种细胞株作为研究对象。通过CCK-8法检测细胞活力,平板克隆和5-乙炔基-2′脱氧尿嘧啶核苷(EdU)实验检测细胞的增殖能力,赫斯特染色(Hoechst)法观察细胞凋亡情况,细胞划痕实验和Transwell实验评估细胞的迁移和侵袭能力,Western blot和定量逆转录PCR(RT-qPCR)法检测磷脂酰肌醇3-激酶/蛋白激酶(PI3K/AKT)信号通路和上皮-间质转化(EMT)的相关蛋白和mRNA的表达差异。结果与A2780细胞相比,A2780/DDP细胞的耐药指数分别约为5.636、5.864、5.695,采用CBG(2、4、6 mg/ml)处理A2780/DDP耐药细胞后,逆转耐药指数分别为1.617、2.570、3.461。CBG呈浓度依赖性地上调细胞凋亡水平、抑制细胞的增殖、迁移和侵袭能力(P<0.05)。Western blot结果显示:与A2780细胞相比,对照组(A2780/DDP)细胞中P-PI3K/PI3K和P-AKT/AKT的蛋白水平相对比值以及N钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、蜗牛蛋白(Snail)的蛋白表达更高,E钙黏蛋白(E-cadherin)蛋白表达更低(t_(P-PI3K/PI3K)=8.115,t_(P-AKT/AKT)=17.62、t_(N-cadherin)=6.126、t_(Vimentin)=4.001、t_(Snail)=17.333、t_(E-cadherin)=4.620,P<0.01);随着CBG剂量升高,耐药细胞中的P-PI3K、P-AKT、N-cadherin、Vimentin、Snail的蛋白表达水平降低,而E-cadherin的蛋白表达量增加(F_(P-PI3K)=268.5、F P-AKT=190.5、F_(N-cadherin)=24.02、F_(Vimentin)=57.65、F_(Snail)=87.24、F_(E-cadherin)=135.8,P<0.05)。RT-qPCR结果显示:随着CBG浓度增加,PI3K、AKT、N-cadherin、Vimentin、Snail的mRNA表达水平随之降低,相反E-cadherin的mRNA表达水平逐渐升高(F PI3K=101.1、F_(AKT)=558.3、F_(N-cadherin)=86.97、F_(Vimentin)=105.9、F_(Snail)=85.71、F_(E-cadherin)=80.96,P<0.01)。结论CBG具有逆转卵巢癌A2780/DDP细胞株顺铂耐药的作用,其机制可能与CBG调控PI3K/AKT信号通路和抑制EMT发生有关。

朊蛋白与p53突变在卵巢浆液性癌组织中表达的相关性及预后意义

目的:探讨卵巢浆液性癌组织中朊蛋白表达水平及其与p53突变的相关性。方法:选取2012年11月10日至2019年10月30日北京大学第三医院妇产科收治的Ⅰ~Ⅳ期卵巢浆液性癌139例,同时选取20例正常卵巢组织、20例卵巢良性肿瘤组织作为对照。免疫组化、p53突变测序法检测朊蛋白表达水平及p53突变类型。结果:朊蛋白在卵巢浆液性癌组织中阳性表达率明显高于卵巢良性肿瘤组织和正常卵巢组织,差异均有统计学意义(59%vs 30%、20%,P分别为0.034、0.003)。卵巢浆液性癌中p53突变率高达84.89%(118/139),主要位于第5外显子(29.66%,35/118)以及DNA结合域的热点突变位点(50.84%,60/118);朊蛋白高表达组中p53构象突变率为71.93%(41/57),显著高于朊蛋白低表达组(45.90%,28/61),差异有统计学意义(P=0.004)。p53构象突变与卵巢浆液性癌淋巴结转移(P=0.005)及耐药(P=0.009)密切相关。p53构象突变患者的平均疾病无进展生存期[(34.99±2.72)月vs(43.01±2.35)月,P=0.036]及平均总生存期[(47.94±3.40)月vs(56.00±2.89)月,P=0.049]均低于p53接触突变患者。结论:卵巢浆液性癌组织中朊蛋白呈高表达,并与p53构象突变相关。p53构象突变与卵巢浆液性癌不良预后相关。

靶向抑制GOT1介导的铁死亡途径逆转食管鳞癌细胞顺铂耐药的机制

目的:探究靶向抑制谷氨酸草酰乙酸转氨酶1(GOT1)是否通过介导铁死亡途径影响食管鳞癌细胞顺铂(DDP)耐药性,并探究分子机制。方法:CCK-8法和细胞集落形成实验检测DDP敏感细胞株Eca-109与DDP耐药细胞株Eca-109/DDP的存活率、集落形成数目,RT-qPCR和Western blot检测Eca-109细胞和Eca-109/DDP细胞中GOT1表达水平差异;将Eca-109/DDP细胞分为对照组、siNC组、siGOT1组、siGOT1+铁死亡抑制剂Ferrostatin-1(Fer-1)组,CCK-8法检测不同浓度DDP处理下各组细胞存活率,细胞集落形成实验检测各组细胞集落形成数目,流式细胞术检测各组细胞凋亡率,DCFH-DA染色法检测各组细胞内活性氧(ROS)水平,比色法检测各组细胞中铁离子(Fe2+)浓度,Western blot检测各组细胞中铁死亡效应因子酰基辅酶A合成酶长链家族成员4 (ACSL4)、谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)的蛋白表达水平。结果:与Eca-109细胞比较,不同浓度DDP处理下Eca-109/DDP细胞存活率增高(P<0.05),细胞集落形成数目增加(P<0.05),细胞中GOT1 mRNA与蛋白相对表达量上调(P<0.05)。与对照组比较,siGOT1组Eca-109/DDP细胞在不同浓度DDP处理下的存活率降低(P<0.05),细胞集落形成数目减少(P<0.05),细胞凋亡率增加(P<0.05),细胞中ROS水平和Fe2+浓度升高(P<0.05),ACSL4蛋白相对表达量上调(P<0.05),GPX4和SLC7A11蛋白相对表达量下调(P<0.05);与siGOT1组比较,siGOT1+Fer-1组Eca-109/DDP细胞在不同浓度DDP处理下的存活率升高(P<0.05),细胞集落形成数目增加(P<0.05),细胞凋亡率减少(P<0.05),同时,细胞中ROS水平和Fe2+浓度降低(P<0.05),ACSL4蛋白相对表达量下调而GPX4和SLC7A11蛋白相对表达量上调(P<0.05)。结论:靶向抑制GOT1能够提高食管鳞癌耐药细胞Eca-109/DDP对DDP的敏感性,促进该耐药细胞凋亡,这一作用与促进铁死亡途径有关。

鼻咽癌顺铂耐药机制的研究进展

鼻咽癌是临床上常见的恶性肿瘤之一。以顺铂为代表药物的化学药物治疗和放射治疗是目前治疗鼻咽癌的主要手段。多发的肿瘤化疗药物耐药现象为鼻咽癌的治疗带来极大困扰。鼻咽癌顺铂耐药机制可以概括为6个方向:增加细胞内药物浓度、减少顺铂的失活物质、增加DNA损伤和抑制损伤修复、促进细胞凋亡、调节肿瘤微环境、抑制胞内自噬。对鼻咽癌细胞在顺铂的耐药性中的研究进展作简要综述,为逆转鼻咽癌顺铂耐药以及寻找天然、安全、有效的化学药物治疗增敏剂的研究提供方向。

肿瘤相关成纤维细胞中微小RNA-214-3p表达对卵巢癌细胞顺铂敏感性的影响

目的探讨肿瘤相关成纤维细胞(CAFs)中微小RNA-214-3p(miR-214-3p)表达对卵巢癌细胞顺铂敏感性的影响及其作用机制。方法选取64例卵巢癌患者作为研究对象,根据化疗后无进展生存期分为铂部分敏感组和铂敏感组,采用实时荧光定量聚合酶链反应(qRT-PCR)检测2组患者卵巢癌组织中miR-214-3p相对表达量,比较不同临床特征患者的2年生存率;原代培养CAFs及正常卵巢成纤维细胞(NFs),采用qRT-PCR、免疫荧光实验检测CAFs和NFs中miR-214-3p、p62蛋白表达;通过CSIOVDB数据库检索SQSTM1基因在不同种类卵巢细胞中的表达水平;向CAFs瞬时转染miR-214-3p mimic(mimic组)和miR-214-3p mimic NC(NC组),将未转染CAFs设为对照组;留取各组细胞培养上清,建立卵巢癌细胞SKOV3与各组CAFs间接共培养模型,采用CCK-8法、DCFH-DA法、qRT-PCR及免疫印迹法分别检测不同培养条件下SKOV3细胞增殖率、顺铂半数抑制浓度(IC 50)、细胞活性氧(ROS)含量和miR-214-3p、顺铂耐药基因CCND1、自噬蛋白p62相对表达量。结果铂部分敏感组患者的miR-214-3p相对表达量低于铂敏感组,差异有统计学意义(P<0.01);不同国际妇产科联盟(FIGO)分期、铂部分敏感情况、miR-214-3p低表达情况患者的2年生存率比较,差异有统计学意义(P<0.01);卵巢CAFs中miR-214-3p相对表达量低于NFs,p62蛋白表达水平高于NFs,差异有统计学意义(P<0.01);CSIOVDB数据库在线分析显示,卵巢癌CAFs中的SQSTM1基因表达水平高于卵巢癌上皮细胞、NFs,差异有统计学意义(P<0.01);相较于与对照组CAFs、NC组CAFs间接共培养的SKOV3细胞,与mimic组CAFs间接共培养的SKOV3细胞的增殖率、顺铂IC 50、ROS含量降低,miR-214-3p相对表达量升高,CCND1mRNA和p62蛋白相对表达量降低,差异有统计学意义(P<0.01)。结论CAFs中miR-214-3p表达与卵巢癌细胞对顺铂的敏感性相关,CAFs中miR-214-3p低表达可促进卵巢癌细胞增殖及ROS介导的自噬,进而降低卵巢癌细胞对顺铂的敏感性。